This project is designed to test if a causal relationship exists between upregulation of Heat Shock Proteins (HSPs) and heat resistance in cells grown in an acid environment. The long term objective is to develop approaches for the identification and the selective killing of tumor cells adapted to growth in low pH environments. The two hypotheses are: 1) Growth of human melanoma cells in an acidic environment induces elevated levels of HSPs, including HSP27 and HSP72, which, in turn, contribute to protection of the cytoplasm and nucleus from heat-induce protein aggregation and disruption, allowing for enhanced synthesis of HSPs and enhanced thermotolerance (TT). Cytoplasmic protection includes protection of the cytoskeleton (CSK) and proton pumps responsible for maintenance of intracellular pH (pHi). 2) The protection of the CSK, pHi homeostasis machinery and the nucleus by HSPs can be overcome by an acute reduction of pHi prior to an during hyperthermia. Specific Aims designed to test the model will examine: i) the interrelationships between levels of HSPs, the phosphorylation of HSP27, the resistance of the CSK to heat-induced collapse & the accumulation of heat-induced nuclear-associated proteins (NAPs), heat-induced synthesis of HSPs and survival in adapted cells; ii) the importance of an intact CSK for realkalinization during hyperthermia following an acid load; and iii) the sensitization of adapted cells by an acute extracellular acidification alone, and by an acute acidification of 0.3 pHe unit combined with inhibitors (DIDS, CNCn, HOE642 and mIBG) that further reduce pHi. HSP levels and phosphorylation of HSP27 will be monitored by Western blot analysis, CSK organization by morphometric and biochemical analyses, NAPs by FCM, and survival by colony formation. Project 2 will be perform all pHi measurements. A translational objective will determine if some of the endpoints will be useful as indicators of the presence of adapted cells in fresh xenograft explants and sections of the xenografts provided by Project 4. This study will contribute to our understanding of the importance of HSP27 and HSP72 for resistance of the CSK and nucleus in adapted cells during hypothermia, and the importance of an intact CSK for efficient function of proton pumps during hyperthermia. Successful accomplishment of the goals may lead to new approaches for identifying and sensitizing acidotic regions of solid tumors to thermoradiotherapy using inhibitors that reduce pHi and/or destabilize the CSK.